. The goals of the proposed studies are the development of RNA expression, trafficking and localization strategies for the enhancement of intracellular ribozyme function in the treatment of HIV-1 infection via gene therapy. The strategies to be tested include: 1) the development of Pol II and Pol III transcription cassettes from sn RNA and tRNA genes for expression of well-defined ribozyme transcripts which localize to specific intracellular compartments; 2) colocalization of ribozymes and HIV-1 RNAs via encapsulation of a tRNA lys3-ribozyme chimera into virions; 3) co- trafficking of ribozyme and target RNAs via 3'untranslated sequences; and 4) facilitation of multiple turnover of substrates by ribozymes mediated by endogenous cellular RNA binding proteins. It is anticipated that a better understanding of the cis-acting sequences directing RNA trafficking within the cell will also be an outcome of these studies. Although the focus of these studies is the hammerhead ribozyme, the lessons learned should be applicable to the hairpin, group 1 and hepatitis delta ribozymes, each of which is being developed for anti- HIV-1 therapeutic applications. In making the choices for ribozyme cleavage targets along the HIV-1 genome, there is the major concern of the genetic variability of the virus resulting in ribozyme resistant variants. It is highly probable in a patient setting that once selective pressure is placed upon the viral population by the use of a ribozyme targeted to any single site, resistant variants will emerge, and perhaps dominate, as has been observed with the nucleoside analogs. It is therefore envisioned that a combination ribozyme expression and targeting strategies coupled with a combination of ribozymes targeted to different sites in the viral genome will prove to be the most effective way of treating HIV-1 infection. By optimizing ribozyme expression, and maximizing the interactions of ribozymes with HIV-1 RNA targets, it should be possible to significantly reduce the infectious viral titer in HIV-1 infected patients. The investigator believes that the studies proposed here will provide the tools for an efficacious ribozyme therapy for the treatment of HIV-1 infection.